Synthesis of 2-alkylcysteine via phase transfer catalysis

ABSTRACT

Non-natural amino acids such as 2-alkylated amino acids allow for the synthesis of a wider variety of peptidal and non-peptidal pharmaceutically active agents. In one embodiment, the present invention provides methods of preparing 2-alkylcysteine derivatives. In another embodiment, the method comprises forming a 2-alkylcysteine derivative from a cysteine derivative in the presence of a phase transfer catalyst. In particular, the present invention provides methods for preparing 2-methylcysteine derivatives. The present invention also discloses a method of preparing a class of iron chelating agents related to desferrithiocin, all of which contain a thiazoline ring. In one aspect, an aryl nitrile or imidate is condensed with cysteine or a 2-alkyl cysteine. The present invention also relates to the preparation of 4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-alkyl-thiazole-4-carboxylic acids, such as 4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-methyl-thiazole-4-carboxylic acid.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/438,757, filed May 15, 2003, which claims the benefit of U.S.Provisional Application Nos. 60/381,012, 60/381,021, 60/380,894,60/380,910, 60/380,880, 60/381,017, 60/380,895, 60/380,903, 60/381,013,60/380,878 and 60/380,909, all of which were filed May 15, 2002. U.S.patent application Ser. No. 10/438,757 also claims the benefit of U.S.Provisional Application No. 60/392,833, filed Jun. 27, 2002. The entireteachings of the above-referenced applications are incorporated hereinby reference.

BACKGROUND OF THE INVENTION

Alpha-amino acids are useful starting materials in the synthesis ofpeptides, as well as non-peptidal, peptidomimetic pharmaceuticallyactive agents. In order to enable the synthesis of a large number ofcompounds from an amino acid precursor, it is advantageous to havenaturally occurring and non-naturally occurring amino acids.Non-naturally occurring amino acids typically differ from natural aminoacids by their stereochemistry (e.g., enantiomers), by the addition ofalkyl groups or other functionalities, or both. At this time, theenantiomers of naturally occurring amino acids are much more expensivethan the naturally occurring amino acids. In addition, there are only alimited number of commercially available amino acids that arefunctionalized or alkylated at the alpha-carbon, and often synthesesinvolve the use of pyrophoric or otherwise hazardous reagents. Moreover,the syntheses are often difficult to scale up to a commercially usefulquantity. Consequently, there is a need for new methodologies ofproducing such non-naturally occurring amino acids.

Non-naturally occurring amino acids of interest include the (R)- and(S)-isomers of 2-methylcysteine, which are used in the design ofpharmaceutically active moieties. Several natural products derived fromthese isomers have been discovered in the past few years. These naturalproducts include desferrithiocin, from Streptomyces antibioticus; aswell as tantazole A, mirabazole C, and thiangazole, all from blue-greenalgae. These compounds have diverse biological activities ranging fromiron chelation to murine solid tumor-selective cytotoxicity toinhibition of HIV-1 infection.

Desferrithiocin, deferiprone, and related compounds represent an advancein iron chelation therapy for subjects suffering from iron overloaddiseases. Present therapeutic agents such as desferroxamine requireparenteral administration and have a very short half-life in the body,so that patient compliance and treatment cost are serious problems forsubjects receiving long-term chelation therapy. Desferrithiocin andrelated compounds are effective when orally administered, therebyreducing patient compliance issues. Unfortunately, (S)-2-methylcysteine,which is a precursor to the more active and/or less toxic forms ofdesferrithiocin and related compounds, remains a synthetic challenge.Therefore, there is a need for novel methods of producing2-methylcysteine at a reasonable cost, and means of isolating thedesired enantiomer.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a method of preparing a2-alkylcysteine derivative represented by Structural Formula (I):

or a salt thereof, wherein

-   -   R₁ is —NH₂; —N(R₅)(R₆); —NHR₇; or —N═R₈; wherein R₅, R₆, R₇, and        R₈ are, independently, a substituted or unsubstituted alkyl        group, a substituted or unsubstituted aromatic group, or a        substituted or unsubstituted heterocyclic group;    -   R₂ and R₃ are, independently, —H, a substituted or unsubstituted        alkyl group, a substituted or unsubstituted aromatic group, or a        substituted or unsubstituted heterocyclic group; and    -   R₄ is a substituted or unsubstituted alkyl group.

In one embodiment, the method comprises reacting a cysteine derivativerepresented by Structural Formula (II):

or a salt thereof, wherein R₁, R₂ and R₃ are defined as above, with acompound having the formula R₄-L, wherein R₄ is defined as above and Lis a leaving group, in the presence of a phase transfer catalyst therebyforming the 2-alkylcysteine derivative represented by Structural Formula(I). Typically, this reaction is carried out in the presence of a base.

The above described methods may additionally comprise the step ofpurifying or ultrapurifying the synthesis products by resolvingenantiomers or diastereomers of the products. The cysteine derivativeformed can be the (R) or (S)-isomer or a mixture thereof. Additionally,the methods can comprise the isolation of the enantiomers of thesynthesis products. In a preferred embodiment, the methods of thepresent invention comprise isolating the (S)-enantiomer of2-alkylcysteine.

The present invention also relates to a method of preparing asubstituted thiazoline carboxylic acid represented by Structural Formula(VII):

or a salt thereof.

In one embodiment, the method comprises:

-   -   (a) reacting, in the presence of a phase transfer catalyst, an        (R)-cysteine derivative represented by Structural Formula        (VIII):        -   or a salt thereof, wherein            -   R₂₀ is —NH₂; —N(R₂₅)(R₂₆); —NHR₂₆; or —N═R₂₇, wherein                R₂₄, R₂₅, R₂₆, and R₂₇ are, independently, a substituted                or unsubstituted alkyl group, a substituted or                unsubstituted aromatic group, or a substituted or                unsubstituted heterocyclic group; and            -   R₂₁, and R₂₂ are, independently, —H, a substituted or                unsubstituted alkyl group, a substituted or                unsubstituted aromatic group, or a substituted or                unsubstituted heterocyclic group;        -   with a compound having the formula CH₃-L, wherein L is a            leaving group, thereby forming a 2-methylcysteine derivative            represented by Structural Formula (IX):        -   or a salt thereof;    -   (b) optionally, purifying the (S)-isomer of the 2-methylcysteine        derivative;    -   (c) reacting the (S)-isomer of the 2-methylcysteine derivative        with acid to form a (S)-2-methylcysteine represented by        Structural Formula (X):    -   (d) coupling the (S)-2-methylcysteine with        2,4-dihydroxybenzonitrile thereby forming the substituted        thiazoline carboxylic acid represented by Structural Formula        (VII).

A further embodiment of the invention includes reacting a cysteine orderivative thereof, including ester and amide derivatives, with abenzonitrile to form a 2-phenyl thiazoline. Suitable cysteines arepreferably substantially enantiomerically pure. Suitable cysteines canalso be substituted at the 2- and 3-positions, preferably alkylated.Preferred cysteines include, separately, the (R)- and (S)-enantiomers of2-methylcysteine, 3,3-dimethylcysteine and 2,3,3-trimethylcysteine,along with esters (e.g., methyl, ethyl) thereof. Benzonitrile arepreferably substituted, such as 2,4-dihydroxybenzonitrile,2-hydroxybenzonitrile, 2,4-dibenyzloxybenzonitrile and2-benzyloxybenzonitrile. The reaction involves reacting the cysteine andthe benzonitrile with a trialkylamine (e.g., trimethylamine,triethylamine, tripropylamine) in an alcoholic solvent (e.g., methanol,ethanol, n-propanol, isopropanol). Preferably, the trialkylamine istriethylamine and the solvent is ethanol. The reaction mixture isadvantageously heated to a temperature from about 50 degrees C. to about150 degrees C., where the mixture refluxes. Also, the reaction ispreferably conducted under an inert atmosphere (e.g., nitrogen, argon,mixtures thereof).

Advantages of the present invention include the facile synthesis of a2-alkylcysteine from cysteine, an inexpensive and readily availablestarting material. 2-Methylcysteine prepared by the method of thepresent invention can be coupled to 2,4-dihydroxybenzonitrile to form4′-hydroxydesazadesferrithiocin, also referred to as4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-methylthiazole-4(S)-carboxylicacid, an iron chelating agent.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides useful and efficient methods of preparing2-alkylcysteine derivatives. The methods include forming a2-alkylcysteine derivative from a cysteine derivative in the presence ofa phase transfer catalyst. Additionally, the present invention relatesto the preparation of4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-alkyl-thiazole-4-carboxylic acid.In particular, the present invention provides methods for preparing2-methylcysteine derivatives as well as4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-methylthiazole-4-carboxylic acid.

In one aspect, the present invention relates to a method of preparing a2-alkylcysteine derivative represented by Structural Formula (I):

or a salt thereof, wherein

-   -   R₁ is —NH₂; —N(R₅)(R₆); —NHR₇; or —N═R₈; wherein R₅, R₆, R₇, and        R₈ are, independently, a substituted or unsubstituted alkyl        group, a substituted or unsubstituted aromatic group, or a        substituted or unsubstituted heterocyclic group;    -   R₂ and R₃ are, independently, —H, a substituted or unsubstituted        alkyl group, a substituted or unsubstituted aromatic group, or a        substituted or unsubstituted heterocyclic group; and    -   R₄ is a substituted or unsubstituted alkyl group.

In one embodiment, the method comprises reacting a cysteine derivativerepresented by Structural Formula (II):

or a salt thereof, wherein R₁, R₂ and R₃ are defined as above, with acompound having the formula R₄-L, wherein R₄ is defined as above and Lis a leaving group, in the presence of a phase transfer catalyst therebyforming the 2-alkylcysteine derivative represented by Structural Formula(I).

In a preferred embodiment, the cysteine derivative reacted is the (R)isomer, represented by Structural Formula (IV):

or a salt thereof, wherein R₁, R₂, and R₃ are as defined above. In anespecially preferred embodiment, the cysteine derivative reacted is aprotected (R)-cysteine and the 2-alkylcysteine derivative thereby formedis a protected 2-methylcysteine. Either the (R) or the (S)-enantiomer ofthe 2-alkylcysteine derivative may be formed in enantiomeric excess.Preferably, the (S)-isomer of a 2-alkylcysteine derivative is formed inenantiomeric excess. More preferably, the (S)-isomer of a protected2-methylcysteine is formed in enantiomeric excess.

The resulting enantiomers of the product can be further resolved andisolated into pure or substantially pure enantiomer components.

Functional groups in compounds of the present invention can be protectedwith protecting groups. Preferably, the cysteine derivative is protectedat any reactive site, for example, at the amino, —SH, and/or carboxylsites of cysteine. A protecting group reduces or eliminates the abilityof a functional group to react under certain conditions. For example, athiol or an alcohol can be protected with an acyl group. Similarly, analcohol or a thiol can be protected by a trityl, a benzyloxymethyl, atetrahydropyranyl or a trimethylsilyl group. An amine can, for example,be protected by an Fmoc group or a Boc group. An acid group can beprotected, for example, by forming an ester or a carboxamide group.Additional protecting groups, methods of adding a protecting group, andmethods of removing a protecting group are taught in “Protective Groupsin Organic Synthesis, 3^(rd) Edition” by Peter G. M. Wuts and TheodoraW. Greene, Wiley-Interscience, 1999, the entire contents of which areincorporated herein by reference.

Preferred protecting groups for acidic nitrogen atoms include formyl;4-toluenesulfonyl; t-butyloxycarbonyl; 2,4-dinitrophenyl;benzyloxymethyl; t-butoxymethyl; 2-chlorobenzyloxy-carbonyl;allyloxycarbonyl; benzyloxycarbonyl (Z); mesitylene-2-sulfonyl;4-methyloxy-2,3,6-trimethyl-benzyenesulfonyl;2,2,5,7,8-pentamethyl-chroma n-6-sulfonyl; 9-xanthenyl; and2,4,6-trimethoxybenzyl.

In one embodiment, R₁ is a protected amino group such as —N═C(Ar)₂wherein each Ar is, independently, a substituted or unsubstituted arylgroup. For example, R₁ can be a benzophenone imine represented byStructural Formula (III):

Preferred protecting groups for acidic sulfur groups include4-methylbenzyl, 3-nitro-2-pyridinesulfenyl; trityl;2,4,6-trimethoxybenzyl; acetamidomethyl; trimethylacetaminomethyl;t-butylsulfonyl; and sulfoxide.

In one embodiment, R₂ is a protecting group protecting the cysteine —SHgroup. For example R₂ can be —C(Ar)₃ wherein each Ar is, independently,a substituted or unsubstituted aryl group. Preferably, R₂ is trityl.

Preferred protecting groups for acidic oxide groups include benzylether; t-butyl ether; benzyl ether; 2,6-dichlorobenzyl ether;2-bromobenzyl ether; and 3,5-dibromobenzyl ether.

Carboxyl groups can be protected, for example, as esters or ascarboxamides. For example, when a carboxyl group is protected as anester, it takes the form of —COOR wherein R is a substituted orunsubstituted C1 to C10 alkyl group, a substituted or unsubstituted upto C30 aryl group, or a substituted or unsubstituted alkyl-aryl groupwherein the alkyl group is C1 to C5 and the aryl group is up to C30.When a carboxyl group is protected as a carboxamide, it takes the formof —CONR′R″ wherein R′ and R″ are, independently, —H, a substituted orunsubstituted C1 to C10 alkyl group, a substituted or unsubstituted upto C30 aryl group, or a substituted or unsubstituted alkyl-aryl groupwherein the alkyl group is C1 to C5 and the aryl group is up to C30.

For example, R₃ can be a carboxyl protecting group such as a substitutedor unsubstituted C1 to C10 alkyl group. In a preferred embodiment, R₃ ist-butyl.

In one incarnation of the present invention, as illustrated below, thecysteine derivative is2(R)-(benzhydrylidene-amino)-3-tritylsulfanyl-propionic acid tert-butylester. 2(R)-(Benzhydrylidene-amino)-3-tritylsulfanyl-propionic acidtert-butyl ester can be formed by the following process: (1)2(R)-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanyl-propionicacid (i.e., (R)-cysteine with Fmoc a protected amino group and with atrityl protected —SH group), is reacted with t-butyl alcohol anddicyclohexyl carbodiimide (DCC) in 4-(dimethylamino)pyridine (DMAP) andtetrahydrofuran (THF) at room temperature to form2(R)-(9H-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanyl-propionicacid tert-butyl ester; (2) the Fmoc group is removed from the2(R)-(9H-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanyl-propionicacid tert-butyl ester using either diethylamine in dichloromethane orpiperidine in dichloromethane to form2(R)-amino-3-tritylsulfanyl-propionic acid tert-butyl ester; and (3) the2(R)-amino-3-tritylsulfanyl-propionic acid tert-butyl ester is reactedwith benzhydrylideneamine in dichloromethane at room temperature to form2(R)-(benzhydrylidene-amino)-3-tritylsulfanyl-propionic acid tert-butylester.

The following sequence illustrates the method described above of forming2(R)-(benzhydrylidene-amino)-3-tritylsulfanyl-propionic acid tert-butylester from2(R)-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanyl-propionicacid:

2(R)-(Benzhydrylidene-amino)-3-tritylsulfanyl-propionic acid tert-butylester

In one embodiment, the cysteine derivative represented by StructuralFormula (II) can be alkylated in the presence of one or more bases, analkylating agent, and a phase transfer catalyst. For example,2-(benzhydrylidene-amino)-3-tritylsulfanyl-propionic acid tert-butylester is reacted with cesium hydroxide monohydrate and excess methyliodide in dichloromethane at about −80° to −60° C. and in the presenceof a phase transfer catalyst. Preferably, the cysteine derivative isalkylated using a phase transfer catalyst such that an enantiomericexcess of either the (R) or (S)-isomer is produced (i.e., the alkylationis stereoselective).

Alkylating agents can have the formula R₄-L, where R₄ is a substitutedor unsubstituted alkyl group and L is a leaving group. Preferred R₄groups include substituted or unsubstituted C1-C4 alkyl groups; methyland benzyl are especially preferred R₄ groups. The leaving group L istypically a weak base. Suitable leaving groups include halogen, tosyl,mesyl, triflyl, brosyl, p-nitrophenyl, and 2,4-dinitrophenyl groups.Halogens include bromine, chlorine, and iodine. Iodine is a preferredleaving group. Suitable amounts of alkylating agent can include about 1to 20, about 2 to 15, about 3 to 10, or, preferably, about 5equivalents, relative to the amount of cysteine derivative.

Preferred bases include alkali or alkaline earth metal hydroxides,alkoxides, amides, or carbonates or their combinations. Available basesinclude potassium t-butoxide, sodium methoxide, sodium ethoxide, sodiumamide, calcium carbonate, cesium carbonate, and the alkali metal saltsof hexamethyl disilazide (HMDS). Preferred bases include potassiumhydroxide, sodium hydroxide, and cesium hydroxide monohydate. Suitableamounts of base include about 5 to 25, about 10 to 20, about 10 to 15,or, preferably, about 10 equivalents, relative to the amount of cysteinederivative.

A phase transfer catalyst functions at the boundary between two solventsor mixtures of solvents, at least one of which is an organic solvent.The organic phase of the process can include any organic solvent whichis substantially inert to the catalyst, reactants and products. Theorganic phase may comprise a combination of two or more solvents.Solvents generally include, but are not limited to, aprotic solventssuch as acetonitrile, acetone, dimethylformamide, dimethyl sulfoxide,tetrahydrofuran, and hexamethylphosphoramide. In a preferred embodiment,the organic phase is comprises dichloromethane.

The alkylation of the cysteine derivative can be performed attemperatures ranging from about −80° C. to about room temperature suchas between about −80° and 0° C. In a preferred embodiment, thealkylation is performed at temperatures of between about −80° and −40°C., for example, at about −60° C.

In one aspect of the present invention, a cinchona-alkaloid derivedphase transfer catalyst is used to alkylate a cysteine derivative. Inone particular embodiment, a cinchona-alkaloid derived phase transfercatalyst is used to stereoselectively alkylate a2-(benzhydrylidene-amino)-3-tritylsulfanyl-propionic acid tert-butylester at the 2-carbon position. The phase transfer catalyst can bederived from cinchonine or from cinchonidine. Use of one of thesecatalysts in the alkylation reaction can yield enantiomeric excesses ofeither the (R) or (S)-enantiomer of the alkylated cysteine derivative,while use of an enantiomer of that catalyst can yield enantiomericexcesses of the other enantiomer of the alkylated cysteine derivative.Thus by selecting the phase transfer catalyst used, one can direct whichenantiomer of the alkylated cysteine derivative will form.

In a preferred embodiment, the phase transfer catalyst used is derivedfrom cinchonidine and is represented by Structural Formula (V):

wherein

-   -   R₉ is a substituted or unsubstituted alkyl group, a substituted        or unsubstituted aromatic group, or a substituted or        unsubstituted heterocyclic group;    -   R₁₀ and R₁₁ are, independently, —H, a substituted or        unsubstituted alkyl group, a substituted or unsubstituted        aromatic group, or a substituted or unsubstituted heterocyclic        group; and    -   X is a halogen.        R₉ can be, for example, substituted or unsubstituted napthyl,        anthracenylmethyl, or benzyl. Preferably, R₉ is        9-anthracenylmethyl as represented by Structural Formula (VI):        R₁₀ can be, for example, substituted or unsubstituted allyl or        benzyl. Preferably, R₁₀ is substituted or unsubstituted allyl.        In another preferred embodiment, R₁₁ is substituted or        unsubstituted ethenyl. In another, X is chlorine or bromine.        Thus the phase transfer catalyst can be represented by        Structural Formula (XI):

Additional examples of phase transfer catalysts suitable for use in thepresent invention are described in U.S. Pat. No. 5,554,753 issued toO'Donnell, et al., the entire teachings of which are incorporated hereinby reference.

The phase transfer catalyst represented by Structural Formula (XI) ispreferably prepared using the following method as described by Corey, etal., in “A Rational Approach to Catalytic Enantioselective EnolateAlkylation Using a Structurally Rigidified and Defined Chiral QuaternaryAmmonium Salt Under Phase Transfer Conditions” (J. Am. Chem. Soc. 119,12414-12415 and Corey Supplemental therein 1-25 (1997)), the entirecontents of which are incorporated by reference herein by reference. Inthat method, cinchonidine, represented by Structural Formula (XII):

is suspended in toluene and 9-(chloromethyl)anthracene, represented byStructural Formula (XIII):

is added. The mixture is stirred at reflux for about 2 hours. Theproduct, N-9-anthracenylmethylcinchonidinium chloride represented byStructural Formula (XIV):

is collected as a light yellow solid. TheN-9-anthracenylmethylcinchonidinium chloride is then suspended indichloromethane. To this suspension is added 50% KOH and allyl bromide.The resulting mixture is then stirred for about 4 hours at about 23° C.The product, O(9)-allyl-N-9-anthracenylmethylcinchonidium bromiderepresented by Structural Formula (XI), is collected as a light orangesolid.

The use of O(9)-allyl-N-9-anthracenylmethylcinchonidium bromide as aphase transfer catalyst is also described in co-pending U.S. patentapplication Ser. Nos. 60/380,903, filed May 15, 2002 and 60/392,833,filed Jun. 27, 2002, the entire contents of which are incorporatedherein by reference.

Examples of other phase transfer catalysts include benzyl triethylammonium chloride, benzyl trimethyl ammonium chloride, benzyl tributylammonium chloride, tetrabutyl ammonium bromide, tetraethyl ammoniumbromide, tetrabutyl ammonium hydrogen sulfate, tetramethyl ammoniumiodide, tetramethyl ammonium chloride, triethylbutyl ammonium bromide,tributyl ethyl ammonium bromide, tributyl methyl ammonium chloride,2-chloroethylamine chloride HCl, bis(2-chloroethyl)amine HCl,2-dimethylaminoethyl chloride HCl, 2-ethylaminoethyl chloride HCl,3-dimethylaminopropyl chloride HCl, methylamine HCl, dimethylamine HCl,trimethylamine HCl, monoethylamine HCl, diethylamine HCl, triethylamineHCl, ethanolamine HCl, diethanolamine HCl, triethanolamine HCl,cyclohexylamine HCl, dicyclohexylamine HCl, cyclohexylamine HCl,diisopropylethylamine HCl, ethylenediamine HCl, aniline HCl, methylsalicylate, ethyl salicylate, butyl salicylate amyl salicylate, isoamylsalicylate, 2-ethylsalicylate, and benzyl salicylate.

In one form of the present invention, the phase transfer catalyst, suchas O(9)-allyl-N-9-anthracenylmethylcinchonidium bromide, is present inan amount of about 0.05 to 0.4 equivalents relative to the amount ofcysteine derivative. Alternatively, the phase transfer catalyst can bepresent between about 0.05 and 0.25 equivalents, between about 0.1 and0.15 equivalents, or, preferably, at about 0.1 equivalents (relative tothe amount of cysteine derivative).

In a preferred embodiment,(R)-2-(benzhydrylidene-amino)-3-tritylsulfanyl-propionic acid tert-butylester is reacted with cesium hydroxide monohydrate and excess methyliodide in dichloromethane at about −60° C. and in the presence ofO(9)-allyl-N-9-anthracenylmethylcinchonidium bromide thereby forming(S)-2-(benzhydrylidene-amino)-2-methyl-3-tritylsulfanyl-propionic acidtert-butyl ester.

Protecting groups, if present, can be removed from the 2-alkylcysteinederivative. Methods of removing a protecting group are well known in theart and taught in “Protective Groups in Organic Synthesis, 3^(rd)Edition” by Wuts and Greene, incorporated by reference above. Forexample,2(S)-(benzhydrylidene-amino)-2-methyl-3-tritylsulfanyl-propionic acidtert-butyl ester can be reacted with acid thereby forming(S)-2-methylcysteine.

The products, either enantiomers or diastereomers, of the above notedsyntheses can be purified or ultrapurified before or after anyprotecting groups are removed. In a preferred embodiment, the2-alkylcysteine derivative (e.g., 2-methylcysteine) is purified byresolution into the (R) and (S)-isomers based on the cysteine 2-carbonposition. For example, the 2-alkylcysteine derivative can be purified orultrapurified using the technique of emulsion crystallization. Emulsioncrystallization may be used to purify acids and functionalizedderivative of acids such as esters and amides. Optionally, theprotective groups are removed after purification to form an unprotected(S)-, or (R)-, 2-alkylcysteine (e.g., 2-methylcysteine) or an (S)-, or(R)-, 2-alkylcysteine derivative.

Alternatively, protective groups are removed from the 2-alkylcysteinederivative to form an unprotected 2-alkylcysteine prior to furtherresolution. For example, protective groups are removed from a protected2-methylcysteine to form an unprotected 2-methylcysteine, theunprotected 2-methylcysteine is resolved into its (R) and (S)-isomers,and an (S)-2-alkylcysteine is isolated. The 2-alkylcysteine can beresolved into its (R) and (S)-isomers using the technique of emulsioncrystallization, or the 2-alkylcysteine can be resolved into itsenantiomers by forming a diastereomeric salt.

Chiral carboxylic acids and their functionalized derivatives, such as2-alkylcysteines and their derivatives, can be purified by emulsioncrystallization, as described in U.S. Pat. No. 5,872,259, 6,383,233 and6,428,583, issued to Reuter, the entire teachings of which areincorporated herein by reference. Briefly, emulsion crystallization is aprocess for separating a desired substance from an aggregate mixture.The process involves forming a three phase system, the first phasecomprising the aggregate mixture, the second phase being liquid andcomprising a transport phase, and the third phase comprising a surfaceupon which the desired substance can crystallize. A chemical potentialexists for crystal growth of the desired substance in the third phase ofthe system, thereby creating a flow of the desired substance from thefirst phase through the second phase to the third phase, where thedesired substance crystallizes and whereby an equilibrium of theactivities of the remaining substances in the aggregate mixture ismaintained between the first phase and the second phase.

In one example of emulsion crystallization, a solution of the racemicmixture is supersaturated (by either cooling, adding a solvent in whichone or more components are sparingly soluble or by evaporation of thesolution). Ultrasonication eventually helps the process of forming anemulsion. The mixture is then seeded with crystals of the desired,optically active acid along with an additional quantity of surfactantand an anti-foaming agent. The desired product usually crystallizes outand can be separated by filtration.

Chiral carboxylic acids also can be purified through further resolutionby forming a diastereomeric salt with the chiral carboxylic acid and achiral amine. Suitable chiral amines include arylalkylamines such as1-alkyl-1-aminoalkanes and 1-aryl-1-aminoalkanes. Examples include(R)-1-phenylethylamine, (S)-1-phenylethylamine, (R)-1-tolylethylamine,(S)-1-tolylethylamine, (R)-1-phenylpropylamine, (S)-1-propylamine,(R)-1-tolylpropylamine, and (S)-1-tolylpropylamine. Preferably,(R)-1-phenylethylamine is used to further resolve the chiral carboxylicacid mixture. Resolution of chiral compounds using diastereomeric saltsis further described in CRC Handbook of Optical Resolutions viaDiastereomeric Salt Formation by David Kozma (CRC Press, 2001),incorporated herein by reference in its entirety.

Once the chiral carboxylic acids or their derivatives have beenpurified, the desired isomer can be isolated. Typically, the (S)-isomeris isolated. For example, protected or unprotected (S)-2-methylcysteine,(S)-2-alkylcysteines, or (S)-2-alkylcysteine derivatives are isolated.Preferably, a protected (S)-2-methylcysteine is isolated.

In a preferred embodiment, a protected (S)-2-methylcysteine is formedand isolated. (S)-2-methylcysteine can then be formed by removing anyprotecting groups present, for example, by treating the protected(S)-2-methylcysteine with acid to remove protecting groups. Cysteine ora 2-alkylcysteine such as (S)-2-methylcysteine can be coupled to asubstituted or unsubstituted aryl nitrile such as a substituted orunsubstituted benzonitrile. Preferably, the substituents on benzonitrilewill not interfere with the coupling reaction. In another preferredembodiment, (S)-2-methylcysteine is coupled to 2,4-dihydroxybenzonitrileto form4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-methylthiazole-4(S)-carboxylicacid (also known as 4′-hydroxydesazadesferrithiocin). In yet anotherembodiment, (S)-2-methylcysteine is coupled to 2-hydroxybenzonitrile toform 4,5-dihydro-2-(2-hydroxyphenyl)-4-methylthiazole-4(S)-carboxylicacid (also known as desazadesferrithiocin).

Typically, coupling of cysteine or a 2-alkylcysteine and a substitutedor unsubstituted benzonitrile includes converting the benzonitrile intoa benzimidate. The benzimidate can be formed, for example, by reactingthe benzonitrile with an alcohol such as methanol, ethanol, n-propanol,or isopropanol in the presence of an acid such as hydrochloric acid. Thebenzimidate is then reacted with the cysteine (or related compound)under basic conditions. Acceptable bases include trimethylamine,triethylamine, triphenylamine, and the like. The reaction between thebenzimidate and the cysteine results in the thiazoline (or4,5-dihydrothiazole) containing product. When forming the benzimidatefrom a hydroxylated benzonitrile (e.g., 2,4-dihydroxybenzonitrile), thehydroxyl groups are advantageously protected (e.g., with a substitutedor unsubstituted alkyl or arylalkyl group such as a benzyl group). Theprotecting groups are subsequently cleaved, typically by catalytichydrogenation.

Suitable benzonitriles and benzimidates for use in the above couplingreaction can be synthesized by methods described in co-pending U.S.patent application Ser. No. 60/381,013, entitled “Synthesis ofBenzonitriles from Substituted Benzoic Acid,” filed May 15, 2002,co-pending U.S. Patent Application No. 60/380,878, entitled “Synthesisof Benzonitriles from Substituted Benzaldehyde,” filed May 15, 2002, andco-pending U.S. patent application Ser. No. 60/380,909, entitled“Synthesis of Benzimidate from Benzoic Acid,” filed May 15, 2002. Theentire contents of these applications are incorporated herein byreference.

The methods of the claimed invention can be used to manufacture otherrelated desferrithiocin analogs and derivatives. Examples of suchanalogs include those described in U.S. Pat. Nos. 5,840,739, 6,083,966,6,159,983, 6,521,652 and 6,525,080, all issued to Bergeron, the contentsof which are incorporated herein by reference. Additional examples canbe found in International Application Nos. PCT/US93/10936, published asWO 94/1137 on May 5, 1994; PCT/US97/04666, published as WO 97/36885 onOct. 9, 1997; and PCT/US99/19691, published as WO 00/12493 on Mar. 9,2000, the entire contents of which are incorporated herein by reference.

An alkyl group is a hydrocarbon in a molecule that is bonded to oneother group in the molecule through a single covalent bond from one ofits carbon atoms. Alkyl groups can be cyclic, branched or unbranched,and/or saturated or unsaturated. Typically, an alkyl group has one toabout 24 carbons atoms, or one to about 12 carbon atoms. Lower alkylgroups have one to four carbon atoms and include methyl, ethyl,n-propyl, iso-propyl, n-butyl, sec-butyl and tert-butyl.

Aromatic (or aryl) groups include carbocyclic aromatic groups such asphenyl, p-tolyl, 1-naphthyl, 2-naphthyl, 1-anthracyl and 2-anthracyl.Aromatic groups also include heteroaromatic groups such as N-imidazolyl,2-imidazole, 2-thienyl, 3-thienyl, 2-furanyl, 3-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 2-pyranyl, 3-pyranyl,3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, 2-pyrazinyl, 2-thiazolyl,4-thiazolyl, 5-thiazolyl, 2-oxazolyl, 4-oxazolyl and 5-oxazolyl.

Aromatic groups also include fused polycyclic aromatic ring systems inwhich a carbocyclic, alicyclic, aromatic ring or heteroaryl ring isfused to one or more other heteroaryl or aryl rings. Examples include2-benzothienyl, 3-benzothienyl, 2-benzofuranyl, 3-benzofuranyl,2-indolyl, 3-indolyl, 2-quinolinyl, 3-quinolinyl, 2-benzothiazole,2-benzooxazole, 2-benzimidazole, 2-quinolinyl, 3-quinolinyl,1-isoquinolinyl, 3-quinolinyl, 1-isoindolyl and 3-isoindolyl.

Suitable substituents for alkyl and aryl groups include —OH, halogen(—Br, —Cl, —I and —F), —O(R′), —O—CO—(R′), —CN, —NO₂, —COOH, ═O, —NH₂,—NH(R′), —N(R′)₂, —COO(R′), —CONH₂, —CONH(R′), —CON(R′)₂, —SH, —S(R′),guanidine, alkyl, and aryl. Each R′ is, independently, an alkyl group oran aromatic group. A substituted alkyl or aryl group can have more thanone substituent.

Also included in the present invention are salts of the disclosedcarboxylic acids. For example, amino acids can also be present in theanionic, or conjugate base, form, in combination with a cation. Suitablecations include alkali metal ions, such as sodium and potassium ions;alkaline earth ions, such as calcium and magnesium ions; andunsubstituted and substituted (primary, secondary, tertiary andquaternary) ammonium ions. Suitable cations also include transitionmetal ions such as manganese, copper, nickel, iron, cobalt, and zinc.Basic groups such as amines can also be protonated with a counter anion,such as hydroxide, halogens (chloride, bromide, and iodide), acetate,formate, citrate, ascorbate, sulfate or phosphate.

EXEMPLIFICATION Example 1 Preparation of the Phase Transfer Catalyst

A cinchonidine derived phase transfer catalyst is prepared as follows.About 4 grams of cinchonidine is suspended in about 40 mL of toluene.About 3 grams of 9-(choloromethyl)anthracene is then added to thesuspension. The mixture is heated to reflux and stirred for about 2hours. Solids are cooled to room temperature, poured onto about 200 mLof diethyl ether and filtered. The product collected isN-9-anthracenylmethylcinchonidinium chloride.

About 5 grams N-9-anthracenylmethylcinchonidinium chloride is thensuspended in about 40 mL dichloromethane. Then about 2.5 mL allylbromide and about 5 mL of 50% KOH (aq) are added to the suspension. Themixture is stirred at about room temperature for about 4 hours. Fiftymilliliters of water is then added to the mixture and the mixture isextracted using three aliquots of dichloromethane. The organic extractsare combined and dried over Na₂SO₄, filtered, and concentrated in vacuo.Recrystalization of the residue from methanol-diethyl ether at −20° C.yields the product, O(9)-allyl-N-9-anthracenylmethylcinchonidiumbromide.

Example 2 Preparation of (S)-2-Methylcysteine

2(R)-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanyl-propionicacid is reacted with t-butyl alcohol and dicyclohexyl carbodiimide (DCC)in 4-(dimethylamino) pyridine (DMAP) and tetrahydrofuran (THF) at roomtemperature to form2(R)-(9H-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanyl-propionicacid tert-butyl ester.

The Fmoc group is removed from the2(R)-(9H-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanyl-propionicacid tert-butyl ester using diethylamine in dichloromethane to form2(R)-amino-3-tritylsulfanyl-propionic acid tert-butyl ester

The 2(R)-amino-3-tritylsulfanyl-propionic acid tert-butyl ester isreacted with benzhydrylideneamine in dichloromethane at room temperatureto form 2(R)-(benzhydrylidene-amino)-3-tritylsulfanyl-propionic acidtert-butyl ester.

A mixture of 2(R)-(benzhydrylidene-amino)-3-tritylsulfanyl-propionicacid tert-butyl ester (one equivalent), about 10 equivalents cesiumhydroxide monohydrate, about 0.1 equivalents ofO(9)-allyl-N-9-anthracenylmethylcinchonidium bromide, and about 0.5 mLdichloromethane is prepared. Excess methyl iodide (about 5 equivalents)is then added dropwise at about −80° C. to the above formed mixture. Themixture is then stirred and allowed to react for about 25-30 hours atabout −60° C. The reacted mixture is then diluted with ether, washedwith water, washed with brine, dried over MgSO₄, filtered andconcentrated in vacuo. The product collected is2-(benzhydrylidene-amino)-2-methyl-3-tritylsulfanyl-propionic acidtert-butyl ester with the (S)-isomer in enantiomeric excess.

The 2-(benzhydrylidene-amino)-2-methyl-3-tritylsulfanyl-propionic acidtert-butyl ester is resolved using emulsion crystallization.2(S)-(benzhydrylidene-amino)-2-methyl-3-tritylsulfanyl-propionic acidtert-butyl ester is then isolated and reacted with excess 5Mhydrochloric acid to form (S)-2-methylcysteine.

Example 3

All compounds were used without further purification. The surfactantsRhodafac RE 610 and Soprophor FL were obtained from Rhóne-Poulenc,Surfynol 465 from Air Products, Synperonic NP 10 from ICI and sodiumlauryl sulfate from Fluka. For agitation a shaking machine was used(Buhler KL Tuttlingen). Purities of the resulting crystals were measuredby using a PolarMonitor polarimeter (IBZ Hannover). Ethanol was used asthe solvent. The total crystal quantity was dissolved in a 1 mL cell at20° C.)

45 mg of (R,R)- and (S,S)-amino acid derivatives were dissolved in 1 mlof a mixture of 20% v/v 2-hexanol, 12% v/v Rhodafac RE 610, 6% v/vSoprophor FL and 62% v/v water by heating to 80° C. in a 5 mL vial.After the organic derivative was completely dissolved the microemulsionwas cooled down to room temperature and agitated using a shaking machine(420 rpm). During two hours no spontaneous crystallization was observed.The mixture was then seeded with two drops of a dilute, finely groundsuspension of pure (S,S)-(−) amino acid or its ester crystals grownunder similar conditions. After 2 hours of agitation the resultingcrystals were filtered off, washed with water and dried in a gentlenitrogen stream.

Example 4

35 mg of R- andS-4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-methylthiazole-4-carboxylic acidwere dissolved in 1 ml of a mixture of 9% N-methyl-pyrrolidone, 9% v/v2-hexanol, 10% v/v Rhodafac RE 610, 5% v/v Soprophor FL and 68% v/vwater by heating to 50° C. in a 5 mL vial. After the product wascompletely dissolved, the microemulsion was cooled down to roomtemperature and agitated with a shaking machine (350 rpm). During twohours, no spontaneous crystallisation was observed. The mixture was thenseeded with two drops of a dilute, finely ground suspension of pureS-product crystals grown under similar conditions. After two hours ofshaking, the resulting crystals were filtered off, washed with water anddried in a gentle nitrogen stream. The procedure yielded 5.4 mg (15.4%)of colorless crystals, with a greater than 90% purity of the Senantiomer.

Example 5

4.00 g (S)-2-methylcysteine hydrochloride (23.3 mmol, 1.0 meq) and 3.14g 2,4-dihydroxy benzonitrile (23.3 mmol, 1.0 meq) were suspended in 40mL ethanol. After degassing this mixture with nitrogen (30 min) 4.95 gtriethylamine (6.8 mL, 48.9 mmol, 2.05 meq) were added. The obtainedsuspension was heated under reflux in an atmosphere of nitrogen for 20hours and then cooled to room temperature. From this suspension ethanolwas evaporated under reduced pressure until an oil (20% of the initialvolume) was obtained. This oil was dissolved in 50 mL water. Thesolution was adjusted to pH 7.5 with 1.20 ml 20% KOH and was extractedtwo times each with 20 mL methyl t-butyl ether (MTBE). The aqueous layerwas separated, adjusted with 20% KOH to pH 11 and again extracted twotimes each with 20 mL MTBE. After separating the aqueous layer the pHwas set with concentrated HCl to 7.5 and traces of MTBE were distilledoff. Then the aqueous solution was acidified with 1.50 ml concentratedHCl to pH 1.5. The product precipitated. This suspension was stirred at4° C. for 1 hour. Then the precipitate was filtered, washed two timeseach with 10 mL water (5° C.) and dried at 45° C. under vacuum. Thereaction yielded 5.17 g (87.6%) of crude4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-methylthiazole-4(S)-carboxylicacid product. ¹H-NMR showed no significant impurity.

Example 6

2,4-Dibenzyloxybenzonitrile (0.121 mol) was dissolved in 5.85 g (0.127mol) ethanol and 19.4 ml 1,2-dimethoxyethane in a double walled reactor.This solution was cooled to −5° C., stirred and saturated with dry HClgas over 5 hours at 0-3° C. The reaction mixture was stirred overnightat 2-4° C. under nitrogen. During this time, a product crystallized. Thewhite crystals were filtered off, washed with 1,2-dimethoxyethane (5°C., three times each with 13 ml) and dried. A total of 30 of theprotected ethyl benzimidate was isolated (Yield 88.4%, purity 98.9%).

The protected ethyl benzimidate described above was dissolved inmethanol to generate a 10% solution and was catalytically hydrogenatedat room temperature using 5% Pd/C as a catalyst. The reaction wascompleted after 8 hours. The solution was filtered and the solventevaporated to yield the deprotected product as an orange-yellow solid.The reaction yielded 19.6 g (94%) of product.

In contrast, the formation of the imidate with 2,4 dihydroxybenzonitrilewas a low yielding process, generating the desired product in only 20%yield and with less than desired purity.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of preparing a substituted thiazoline carboxylic acidrepresented by Structural Formula (VII):

or a salt thereof, the method comprising: (a) reacting, in the presenceof a phase transfer catalyst, an (R)-cysteine derivative represented byStructural Formula (VIII):

or a salt thereof, wherein R₂₀ is —NH₂; —N(R₂₅)(R₂₆); —NHR₂₆; or —N═R₂₇,wherein R₂₄, R₂₅, R₂₆, and R₂₇ are, independently, a substituted orunsubstituted alkyl group, a substituted or unsubstituted aromaticgroup, or a substituted or unsubstituted heterocyclic group; and R₂₁ andR₂₂ are, independently, —H, a substituted or unsubstituted alkyl group,a substituted or unsubstituted aromatic group, or a substituted orunsubstituted heterocyclic group; with a compound having the formulaCH₃-L, wherein L is a leaving group, thereby forming a 2-methylcysteinederivative represented by Structural Formula (IX):

or a salt thereof; (b) optionally, purifying the (S)-isomer of the2-methylcysteine derivative; (c) reacting the (S)-isomer of the2-methylcysteine derivative with acid to form (S)-2-methylcysteinerepresented by Structural Formula (X):

(d) coupling the (S)-2-methylcysteine with 2,4-dihydroxybenzonitrilethereby forming the substituted thiazoline carboxylic acid representedby Structural Formula (VII).
 2. The method of claim 1 wherein the phasetransfer catalyst is a compound represented by Structural Formula (V):

wherein R₉ is a substituted or unsubstituted alkyl group, a substitutedor unsubstituted aromatic group, or a substituted or unsubstitutedheterocyclic group; R₁₀ and R₁₁ are, independently, —H, a substituted orunsubstituted alkyl group, a substituted or unsubstituted aromaticgroup, or a substituted or unsubstituted heterocyclic group; and X is ahalogen.
 3. The method of claim 2 wherein R₉ is 9-anthracenylmethyl,represented by the Structural Formula (VI):


4. The method of claim 2 wherein R10 is substituted or unsubstitutedallyl.
 5. The method of claim 2 wherein R11 is substituted orunsubstituted ethenyl.
 6. The method of claim 2 wherein X is chlorine.7. The method of claim 1, wherein the phase transfer catalyst isselected from the group consisting of benzyl triethyl ammonium chloride,benzyl trimethyl ammonium chloride, benzyl tributyl ammonium chloride,tetrabutyl ammonium bromide, tetraethyl ammonium bromide, tetrabutylammonium hydrogen sulfate, tetramethyl ammonium iodide, tetramethylammonium chloride, triethylbutyl ammonium bromide, tributyl ethylammonium bromide, tributyl methyl ammonium chloride, 2-chloroethylaminechloride HCl, bis(2-chloroethyl)amine HCl, 2-dimethylaminoethyl chlorideHCl, 2-ethylaminoethyl chloride HCl, 3-dimethylaminopropyl chloride HCl,methylamine HCl, dimethylamine HCl, trimethylamine HCl, monoethylamineHCl, diethylamine HCl, triethylamine HCl, ethanolamine HCl,diethanolamine HCl, triethanolamine HCl, cyclohexylamine HCl,dicyclohexylamine HCl, cyclohexylamine HCl, diisopropylethylamine HCl,ethylenediamine HCl, aniline HCl, methyl salicylate, ethyl salicylate,butyl salicylate amyl salicylate, isoamyl salicylate, 2-ethylsalicylate,and benzyl salicylate.
 8. A method of preparing a 2-alkylcysteinederivative represented by Structural Formula (I):

or a salt thereof, wherein R₁ is —NH₂; —N(R₅)(R₆); —NHR₇; or —N═R₈;wherein R₅, R₆, R₇, and R₈ are, independently, a substituted orunsubstituted alkyl group, a substituted or unsubstituted aromaticgroup, or a substituted or unsubstituted heterocyclic group; R₂ and R₃are, independently, —H, a substituted or unsubstituted alkyl group, asubstituted or unsubstituted aromatic group, or a substituted orunsubstituted heterocyclic group; and R₄ is a substituted orunsubstituted alkyl group; the method comprising, reacting, in thepresence of a phase transfer catalyst, a cysteine derivative representedby Structural Formula (II):

or a salt thereof, wherein R₁, R₂ and R₃ are defined as above, with acompound having the formula R₄-L, wherein R₄ is defined as above and Lis a leaving group thereby forming the 2-alkylcysteine derivativerepresented by Structural Formula (I), wherein the phase transfercatalyst is selected from the group consisting of benzyl triethylammonium chloride, benzyl trimethyl ammonium chloride, benzyl tributylammonium chloride, tetrabutyl ammonium bromide, tetraethyl ammoniumbromide, tetrabutyl ammonium hydrogen sulfate, tetramethyl ammoniumiodide, tetramethyl ammonium chloride, triethylbutyl ammonium bromide,tributyl ethyl ammonium bromide, tributyl methyl ammonium chloride,2-chloroethylamine chloride HCl, bis(2-chloroethyl)amine HCl,2-dimethylaminoethyl chloride HCl, 2-ethylaminoethyl chloride HCl,3-dimethylaminopropyl chloride HCl, methylamine HCl, dimethylamine HCl,trimethylamine HCl, monoethylamine HCl, diethylamine HCl, triethylamineHCl, ethanolamine HCl, diethanolamine HCl, triethanolamine HCl,cyclohexylamine HCl, dicyclohexylamine HCl, cyclohexylamine HCl,diisopropylethylamine HCl, ethylenediamine HCl, aniline HCl, methylsalicylate, ethyl salicylate, butyl salicylate amyl salicylate, isoamylsalicylate, 2-ethylsalicylate, and benzyl salicylate.
 9. The method ofclaim 8, wherein R₁ is —N═C(Ar)₂ wherein each Ar is, independently, asubstituted or unsubstituted aryl group.
 10. The method of claim 9,wherein R₁ is a benzophenone imine represented by Structural Formula(III):


11. The method of claim 8, wherein R₂ is —C(Ar)₃ wherein each Ar is,independently, a substituted or unsubstituted aryl group.
 12. The methodof claim 11, wherein R₂ is trityl.
 13. The method of claim 8, wherein R₃is a substituted or unsubstituted C1 to C10 alkyl group.
 14. The methodof claim 13, wherein R₃ is t-butyl.
 15. The method of claim 8, whereinthe cysteine derivative is the (R) isomer, represented by StructuralFormula (IV):


16. The method of claim 8, wherein R₄ is a substituted or unsubstitutedC1 to C4 alkyl group.
 17. The method of claim 16, wherein R₄ is methyl.18. The method of claim 8, wherein the cysteine derivative is protectedat any acidic nitrogen, oxygen, or sulfur atoms.
 19. The method of claim8, further comprising the step of resolving the enantiomers of the2-alkylcysteine derivative.
 20. The method of claim 19, wherein the(S)-isomer of the 2-alkylcysteine derivative is isolated.